Compositions and methods for detection of BK virus

ABSTRACT

Methods for the rapid detection of the presence or absence of BK virus in a biological or non-biological sample are described. The methods can include performing an amplifying step, a hybridizing step, and a detecting step. Furthermore, primers and probes targeting BK virus and kits are provided that are designed for the detection of BK virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation patent application of U.S. patentapplication Ser. No. 15/726,732, filed Oct. 6, 2017, which claims thebenefit of priority of U.S. Provisional Patent Application No.62/419,853, filed Nov. 10, 2016, both of which are incorporated hereinby reference in their entireties.

SEQUENCE LISTING

The instant patent application contains a Sequence Listing, which hasbeen submitted electronically in both computer readable format (CRF) andwritten (paper) form, and is hereby incorporated by reference in itsentirety. The written (paper) form copy, created Aug. 27, 2020, is named“33881-US1_Sequence_Listing,” and is 26,610 bytes in size.

FIELD OF THE INVENTION

The present disclosure relates to the field of in vitro viraldiagnostics. Within this field, the present invention concerns theamplification and detection of a target nucleic acid that may be presentin a sample and particularly, the amplification, detection, andquantitation of a target nucleic acid comprising sequence variationsand/or individual mutations of BK virus, using primers and probes. Theinvention further provides reaction mixtures and kits containing primersand probes for amplification and detection of BK virus.

BACKGROUND OF THE INVENTION

BK virus, a member of the Polyomaviridae family, was first isolated in1971 from the urine of a renal transplant recipient with uretericstenosis, having the initials “B.K.” The Polyomaviridae family includesJohn Cunningham virus (JCV) and simian virus SV40. Although BK virusinfections are widespread, infected individuals are usually asymptomaticor exhibit only mild symptoms (e.g., respiratory infection or fever).The BK virus is a circular, double-stranded DNA virus. Its genomeencodes three capsid structural proteins (i.e., viral capsid protein 1(VP1), VP2, and VP3), as well as the large T and small t antigens.

After primary infection, the virus typically establishes latency in theuroepithelium and renal tubular epithelial cells. It is believed that upto 80% of the population contains a latent form of this virus. Thesymptoms of a BK virus infection for individuals who areimmunosuppressed and/or immunocompromised, are significantly moresevere, for example, in the setting of an organ transplant. In suchcase, clinical manifestations can include renal dysfunction and thepresence of renal tubular cells and inflammatory cells in urine. Inparticular, in the setting of immunosuppression and/or immunocompromise,the virus reactivates and replicates, triggering a cascade of eventsstarting with tubular cell lysis and viruria. The virus then multipliesin the interstitium and crosses into the peritubular capillaries,causing viremia and eventually invading the allograft, leading tovarious tubulointerstitial lesions and BK nephropathy (BKVN). The BKvirus contributes significantly to the increase in probability of graftloss. BK virus is a common post-transplant opportunistic viralinfection, affecting roughly 15% of renal plant recipients in the firstpost-transplant year. If unaddressed, BK nephropathy will progress toallograft failure.

Though being immunosuppressed and/or immunocompromised remains theprimary risk factor for BK virus infection, other risk factors includemale gender, older recipient age, prior rejection episodes, degree ofhuman leukocyte antigen mismatching, prolonged cold ischemia time, BKserostatus, and ureteral stent placement. Treatment options forsymptomatic BK virus-infected individuals are limited and there is noeffective prophylaxis. The cornerstone of treatment is simply reductionof immunosuppression, which increases the risk of allograft rejection.Anti-viral drugs are also employed, but with inconsistent results. BKvirus is now recognized as a chief cause of interstitial nephritis andallograft failure in renal transplant recipients.

BK virus infection is diagnosed by a BK virus blood test or a urine testfor decoy cells. The presence of decoy sells is a sensitive measure, buthas a low positive predictive value (29%) for the diagnosis of BKnephropathy (see, Mbianda, et al. Journal of Clinical Virology 71:59-62(2015). Quantification of viral load in the plasma and urine with eitherviral DNA or mRNA has also been used to diagnose BK nephropathy.However, a transplant kidney biopsy remains the gold standard fordiagnosing BK virus nephropathy.

In the field of molecular diagnostics, the amplification and detectionof nucleic acids is of considerable significance. Such methods can beemployed to detect any number of microorganisms, such as viruses andbacteria. The most prominent and widely-used amplification technique isthe Polymerase Chain Reaction (PCR). Other amplification techniquesinclude Ligase Chain Reaction, Polymerase Ligase Chain Reaction,Gap-LCR, Repair Chain Reaction, 3 SR, NASBA, Strand DisplacementAmplification (SDA), Transcription Mediated Amplification (TMA), andQβ-amplification.

Automated systems for PCR-based analysis often make use of a real-timedetection of product amplification during the PCR process in the samereaction vessel. Key to such methods is the use of modifiedoligonucleotides carrying reporter groups or labels.

An estimated 80% of the population harbors the BK virus in a latentstate, many of them unknowingly so, because the symptoms of a BK virusinfection are so mild or non-existent. The gold standard for diagnosingBK nephropathy is a transplant kidney biopsy, which is a time-consuming,invasive, and laborious procedure. Therefore, there is a need in the artfor a quick, reliable, specific, and sensitive method for detecting andquantifying the presence of BK virus in a biological sample.

SUMMARY OF THE INVENTION

Certain embodiments in the present disclosure relate to methods for therapid detection of the presence or absence of BK virus in a biologicalor non-biological sample, for example, multiplex detection andquantitating of BK virus by real-time polymerase chain reaction (PCR) ina single test tube or vessel. Embodiments include methods of detectionof BK virus comprising performing at least one cycling step, which mayinclude an amplifying step and a hybridizing step. Furthermore,embodiments include primers, probes, and kits that are designed for thedetection of BK virus in a single tube or vessel.

One embodiment of the claimed invention is directed to a method ofdetecting BK Virus in a sample, the method comprising: (a) performing anamplifying step comprising contacting the sample with one or more set ofprimers to produce an amplification product, if a target nucleic acid ofBK Virus is present in the sample; (b) performing a hybridization stepcomprising contacting the amplification product, if a target nucleicacid is present in the sample, with one or more probes; and (c)detecting the presence or absence of the amplification product, whereinthe presence of the amplification product is indicative of the presenceof BK Virus in the sample, and wherein the absence of the amplificationproduct is indicative of the absence of BK Virus in the sample; whereinthe one or more set of primers comprise at least two primers selectedfrom a group consisting of SEQ ID NOs:1, 2, 4, 5, 6, 7, 9, and 10, or acomplement thereof; and wherein the one or more probes are selected froma group consisting of SEQ ID NOs:3, 8, and 11, or a complement thereof.In a related embodiment, the one or more set of primers comprises twoprimers consisting of SEQ ID NOs:1 and 2, or a complement thereof, andthe one or more probes comprises a probe consisting of SEQ ID NO:3, or acomplement thereof. In a related embodiment, the one or more set ofprimers comprises two primers consisting of SEQ ID NOs:4 and 5, or acomplement thereof, and the one or more probes comprises a probeconsisting of SEQ ID NO:3, or a complement thereof. In a relatedembodiment, the one or more set of primers comprises two primersconsisting of SEQ ID NOs:6 and 7, or a complement thereof, and the oneor more probes comprises a probe consisting of SEQ ID NO:8, or acomplement thereof. In a related embodiment, the one or more set ofprimers comprises two primers consisting of SEQ ID NOs:9 and 10, or acomplement thereof, and the one or more probes comprises a probeconsisting of SEQ ID NO:11, or a complement thereof. In otherembodiment, the hybridization step comprises contacting theamplification product with the probe that is labeled with a donorfluorescent moiety and a corresponding acceptor moiety; the detectingstep comprises detecting the presence or absence of fluorescenceresonance energy transfer (FRET) between the donor fluorescent moietyand the acceptor moiety of the probe, and the presence or absence offluorescence is indicative of the presence of absence of BK Virus in thesample. In another embodiment, the amplification step comprises apolymerase enzyme having 5′ to 3′ nuclease activity. In anotherembodiment, the acceptor moiety is a quencher, such as BlackHoleQuencher™-2 (BHQ-2). In another embodiment, the donor fluorescent moietyis HEX. In another embodiment, the sample is a biological sample, whichincludes, but is not limited to, blood, plasma, or urine. In yet anotherembodiment, the method further comprises detecting, in in parallel, asecond target nucleic acid from one or more other microorganisms. In arelated embodiment, the one or more other microorganisms is a bacteria.In a related embodiment, the one or more other microorganisms is avirus, including, but not limited to, Hepatitis A Virus (HAV), HepatitisB Virus (HBV), Hepatitis C Virus (HCV), Hepatitis E Virus (HEV), HumanImmunodeficiency Virus (HIV), West Nile Virus (WNV), JapaneseEncephalitis Virus (JEV), Zika Virus, Dengue Fever Virus, St. LouisEncephalitis Virus (SLEV), and/or Chikungunya Virus.

Another embodiment of the claimed invention is directed a kit fordetecting a nucleic acid of BK Virus comprising: (a) a set of primerscomprising at least two primers selected from the a group consisting ofSEQ ID NOs:1, 2, 4, 5, 6, 7, 9, and 10, or a complement thereof; and (b)one or more fluorescently detectably labeled probes selected from agroup consisting of SEQ ID NOs:3, 8, and 11, or a complement thereof,wherein the fluorescently detectably labeled probe is configured tohybridize to an amplicon generated by the at least two primers. In arelated embodiment, the set of primers comprises two primers consistingof SEQ ID NOs:1 and 2, or a complement thereof, and the one or moreprobes comprises a probe consisting of SEQ ID NO:3, or a complementthereof. In a related embodiment, the set of primers comprises twoprimers consisting of SEQ ID NOs:4 and 5, or a complement thereof, andthe one or more probes comprises a probe consisting of SEQ ID NO:3, or acomplement thereof. In a related embodiment, the set of primerscomprises two primers consisting of SEQ ID NOs:6 and 7, or a complementthereof, and the one or more probes comprises a probe consisting of SEQID NO:8, or a complement thereof. In a related embodiment, the set ofprimers comprises two primers consisting of SEQ ID NOs:9 and 10, or acomplement thereof, and the one or more probes comprises a probeconsisting of SEQ ID NO:11, or a complement thereof. In anotherembodiment, the detectably labeled oligonucleotide sequence comprises adonor fluorescent moiety and a corresponding acceptor moiety. In oneembodiment, the acceptor moiety is a quencher, such as BlackHoleQuencher™-2 (BHQ-2). In another embodiment, the donor fluorescent moietyis HEX. In one embodiment, the sample is a biological sample, including,but not limited to, blood, plasma, or urine. In another embodiment, thekit further comprises primers and probes for the amplification anddetection of a second target from one or more other microorganisms. Inone embodiment, the one or more microorganisms is a bacteria. In oneembodiment, the one or more other microorganisms is a virus, including,but not limited to, Hepatitis A Virus (HAV), Hepatitis B Virus (HBV),Hepatitis C Virus (HCV), Hepatitis E Virus (HEV), Human ImmunodeficiencyVirus (HIV), West Nile Virus (WNV), Japanese Encephalitis Virus (JEV),Zika Virus, Dengue Fever Virus, St. Louis Encephalitis Virus (SLEV),and/or Chikungunya Virus. In yet another embodiment, the kit furthercomprises nucleoside triphosphates, nucleic acid polymerase, and buffersnecessary for the function of the nucleic acid polymerase. In anotherembodiment, the at least one of either the at least two primers or thefluorescently detectable labeled probe comprises at least one modifiednucleotide.

Another embodiment of the claimed invention is directed to a set ofprimers and one or more probes for the detection of BK Virus in asample, wherein the set of primers comprises at least two primersselected from a group consisting of SEQ ID NOs:1, 2, 4, 5, 6, 7, 9, and10, or a complement thereof; and the one or more probes are selectedfrom a group consisting of SEQ ID NOs:3, 8, and 11, or a complementthereof. In a related embodiment, the set of primers comprises twoprimers consisting of SEQ ID NOs:1 and 2, or a complement thereof, andthe one or more probes comprises a probe consisting of SEQ ID NO:3, or acomplement thereof. In a related embodiment, the set of primerscomprises two primers consisting of SEQ ID NOs:4 and 5, or a complementthereof, and the one or more probes comprises a probe consisting of SEQID NO:3, or a complement thereof. In a related embodiment, the set ofprimers comprises two primers consisting of SEQ ID NOs:6 and 7, or acomplement thereof, and the one or more probes comprises a probeconsisting of SEQ ID NO:8, or a complement thereof. In a relatedembodiment, the set of primers comprises two primers consisting of SEQID NOs:9 and 10, or a complement thereof, and the one or more probescomprises a probe consisting of SEQ ID NO:11, or a complement thereof.In another embodiment, an amplifying step is performed comprisingcontacting a sample with the set of primers to produce an amplificationproduct if a nucleic acid is present in the sample; performing ahybridization step comprising contacting the amplification product withthe one or more probes; and detecting the presence or absence of theamplification product, wherein the presence of the amplification productis indicative of the presence of BK Virus in the sample and wherein theabsence of the amplification product is indicative of the absence of BKVirus in the sample. In one embodiment, the hybridization step comprisescontacting the amplification product with the probe that is labeled witha donor fluorescent moiety and a corresponding acceptor moiety; and thedetecting step comprises detecting the presence or absence of FRETbetween the donor fluorescent moiety and the acceptor moiety of theprobe, wherein the presence or absence of fluorescence is indicative ofthe presence or absence of BK Virus in the sample. In one embodiment,the amplification step comprises a polymerase enzyme having 5′ to 3′nuclease activity. In another embodiment, the acceptor moiety is aquencher, such as BlackHole Quencher™-2 (BHQ-2). In one embodiment, thedonor fluorescent moiety is HEX. In one embodiment, the sample is abiological sample, including, but not limited to blood, plasma, orurine.

Other embodiments provide an oligonucleotide comprising or consisting ofa sequence of nucleotides selected from SEQ ID NOs:1-11, or a complementthereof, which oligonucleotide has 100 or fewer nucleotides. In anotherembodiment, the present disclosure provides an oligonucleotide thatincludes a nucleic acid having at least 70% sequence identity (e.g., atleast 75%, 80%, 85%, 90% or 95%, etc.) to one of SEQ ID NOs:1-11, or acomplement thereof, which oligonucleotide has 100 or fewer nucleotides.Generally, these oligonucleotides may be primer nucleic acids, probenucleic acids, or the like in these embodiments. In certain of theseembodiments, the oligonucleotides have 40 or fewer nucleotides (e.g., 35or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides,20 or fewer nucleotides, 15 or fewer nucleotides, etc.) In someembodiments, the oligonucleotides comprise at least one modifiednucleotide, e.g., to alter nucleic acid hybridization stability relativeto unmodified nucleotides. Optionally, the oligonucleotides comprise atleast one label and optionally at least one quencher moiety. In someembodiments, the oligonucleotides include at least one conservativelymodified variation. “Conservatively modified variations” or, simply,“conservative variations” of a particular nucleic acid sequence refersto those nucleic acids, which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skill inthe art will recognize that individual substitutions, deletions oradditions which alter, add or delete a single nucleotide or a smallpercentage of nucleotides (typically less than 5%, more typically lessthan 4%, 2% or 1%) in an encoded sequence are “conservatively modifiedvariations” where the alterations result in the deletion of an aminoacid, addition of an amino acid, or substitution of an amino acid with achemically similar amino acid.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ nuclease activity. Thus, the donor fluorescent moiety and theacceptor moiety, e.g., a quencher, may be within no more than 5 to 20nucleotides (e.g., within 8 or 10 nucleotides) of each other along thelength of the probe. In another aspect, the probe includes a nucleicacid sequence that permits secondary structure formation. Such secondarystructure formation may result in spatial proximity between the firstand second fluorescent moiety. According to this method, the secondfluorescent moiety on the probe can be a quencher.

The present disclosure also provides for methods of detecting thepresence or absence of BK virus or BK virus nucleic acid, in abiological sample from an individual. These methods can be employed todetect the presence or absence of BK virus nucleic acid in plasma, foruse in blood screening and diagnostic testing. Additionally, the sametest may be used by someone experienced in the art to assess urine andother sample types to detect and/or quantitate BK virus nucleic acid.Such methods generally include performing at least one cycling step,which includes an amplifying step and a dye-binding step. Typically, theamplifying step includes contacting the sample with a plurality of pairsof oligonucleotide primers to produce one or more amplification productsif a nucleic acid molecule is present in the sample, and the dye-bindingstep includes contacting the amplification product with adouble-stranded DNA binding dye. Such methods also include detecting thepresence or absence of binding of the double-stranded DNA binding dyeinto the amplification product, wherein the presence of binding isindicative of the presence of BK virus nucleic acid in the sample, andwherein the absence of binding is indicative of the absence of BK virusnucleic acid in the sample. A representative double-stranded DNA bindingdye is ethidium bromide. Other nucleic acid-binding dyes include DAPI,Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes suchas YO-YO® and SYBR® Green. In addition, such methods also can includedetermining the melting temperature between the amplification productand the double-stranded DNA binding dye, wherein the melting temperatureconfirms the presence or absence of BK virus nucleic acid nucleic acid.

In a further embodiment, a kit for detecting and/or quantitating one ormore nucleic acids of BK virus is provided. The kit can include one ormore sets of primers specific for amplification of the gene target; andone or more detectable oligonucleotide probes specific for detection ofthe amplification products.

In one aspect, the kit can include probes already labeled with donor andcorresponding acceptor moieties, e.g., another fluorescent moiety or adark quencher, or can include fluorophoric moieties for labeling theprobes. The kit can also include nucleoside triphosphates, nucleic acidpolymerase, and buffers necessary for the function of the nucleic acidpolymerase. The kit can also include a package insert and instructionsfor using the primers, probes, and fluorophoric moieties to detect thepresence or absence of BK virus nucleic acid in a sample.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present subject matter, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows real-time PCR growth curves showing the detection of BKvirus genomic DNA samples (4×10⁵ genomes/μl) by primers (SEQ ID NOs:4and 5) and probe (SEQ ID NO:3) specific for BK virus.

FIG. 2 shows real-time PCR growth curves showing the specific detectionof BK virus genomic DNA samples (4×10⁵ genomes/μl) by primers (SEQ IDNOs:4 and 5) and probe (SEQ ID NO:3). BK virus primers and probes arenot cross-reactive with genomic DNA from other viral DNA samples fromEpstein Barr Virus (EBV), Herpes Simplex Virus-1 (HSV-1), HSV-2, andCytomegalovirus (CMV), tested at 1×10⁸ copies/μl. No signal was observedfor the genomic DNA samples other than BK virus.

FIG. 3 shows real-time PCR growth curves showing the sensitivity ofdetection of BK virus genomic DNA samples by primers (SEQ ID NOs:1 and2) and probe (SEQ ID NO:3). A dilution panel was employed with DNAsamples at the following varying concentrations: 1×10¹ genomes/μl, 1×10²genomes/μl, 1×10³ genomes/μl, 1×10⁴ genomes/μl, 1×10⁵ genomes/μl, and4×10⁵ genomes/μl. Results demonstrate the sensitivity of the primers andprobes, showing the ability to detect BK virus DNA at levels as low as1×10¹ genomes/μl.

FIG. 4 shows real-time PCR growth curves showing the sensitivity ofdetection of BK virus genomic DNA samples by primers (SEQ ID NOs:6 and7) and probe (SEQ ID NO:8). A dilution panel was employed with DNAsamples at the following varying concentrations: 1×10¹ genomes/μl, 1×10²genomes/μl, and 1×10³ genomes/μl. Results demonstrate the sensitivity ofthe primers and probes, showing the ability to detect BK virus DNA atlevels as low as 1×10¹ genomes/μl

FIG. 5 shows real-time PCR growth curves showing the sensitivity ofdetection of BK virus genomic DNA samples by primers (SEQ ID NOs:9 and10) and probe (SEQ ID NO:11). A dilution panel was employed with DNAsamples at the following varying concentrations: 1×10¹ genomes/μl, 1×10²genomes/μl, and 1×10³ genomes/μl. Results demonstrate the sensitivity ofthe primers and probes, showing the ability to detect BK virus DNA atlevels as low as 1×10¹ genomes/μl.

FIG. 6 shows real-time PCR growth curves showing the multiplex detectionof BK virus genomic DNA samples using two different sets ofoligonucleotide sets (SEQ ID NOs:3-5 and SEQ ID NOs:6-8). Resultsdemonstrate that two sets of oligonucleotides (SEQ ID NOs:3-5 and SEQ IDNOs:6-8) amplify and detect BK virus in a multiplex setting.

FIG. 7 shows real-time PCR growth curves showing the multiplex detectionof BK virus genomic DNA samples using two different sets ofoligonucleotide sets (SEQ ID NOs:3-5 and SEQ ID NOs:9-11). Resultsdemonstrate that two sets of oligonucleotides (SEQ ID NOs:3-5 and SEQ IDNOs:9-11) amplify and detect BK virus in a multiplex setting.

DETAILED DESCRIPTION OF THE INVENTION

Diagnosis of BK virus infection by nucleic acid amplification provides amethod for rapidly, accurately, reliably, specifically, and sensitivelydetecting and/or quantitating the BK viral infection. A real-time PCRassay for detecting and/or quantitating BK virus in a non-biological orbiological sample is described herein. Primers and probes for detectingand/or quantitating BK virus are provided, as are articles ofmanufacture or kits containing such primers and probes. The increasedspecificity and sensitivity of real-time PCR for detection of BK viruscompared to other methods, as well as the improved features of real-timePCR including sample containment and real-time detection andquantitating of the amplified product, make feasible the implementationof this technology for routine diagnosis of BK virus infections in theclinical laboratory. Additionally, this technology may be employed forblood screening as well as for prognosis. This BK virus detection assaymay also be multiplexed with other assays for the detection of othernucleic acids, e.g., other bacteria and/or viruses, in parallel.

The present disclosure includes oligonucleotide primers and fluorescentlabeled hydrolysis probes that hybridize to the BK virus genome, inorder to specifically identify BK virus using, e.g., TaqMan®amplification and detection technology.

The disclosed methods may include performing at least one cycling stepthat includes amplifying one or more portions of the nucleic acidmolecule gene target from a sample using one or more pairs of primers.“BK primer(s)” as used herein refer to oligonucleotide primers thatspecifically anneal to nucleic acid sequences found in the BK virusgenome, and initiate DNA synthesis therefrom under appropriateconditions producing the respective amplification products. Examples ofnucleic acid sequences found in the BK virus genome, include nucleicacids within viral capsid protein region of the BK virus genome, such asthe VP2 region. Each of the discussed BK virus primers anneals to atarget such that at least a portion of each amplification productcontains nucleic acid sequence corresponding to the target. The one ormore amplification products are produced provided that one or morenucleic acid is present in the sample, thus the presence of the one ormore amplification products is indicative of the presence of BK virus inthe sample. The amplification product should contain the nucleic acidsequences that are complementary to one or more detectable probes for BKvirus. “BK virus probe(s)” as used herein refer to oligonucleotideprobes that specifically anneal to nucleic acid sequences found in theBK virus genome. Each cycling step includes an amplification step, ahybridization step, and a detection step, in which the sample iscontacted with the one or more detectable BK virus probes for detectionof the presence or absence of BK virus in the sample.

As used herein, the term “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid molecule (e.g., nucleic acidmolecules from the BK virus genome). Amplifying a nucleic acid moleculetypically includes denaturing the template nucleic acid, annealingprimers to the template nucleic acid at a temperature that is below themelting temperatures of the primers, and enzymatically elongating fromthe primers to generate an amplification product. Amplificationtypically requires the presence of deoxyribonucleoside triphosphates, aDNA polymerase enzyme (e.g., Platinum® Taq) and an appropriate bufferand/or co-factors for optimal activity of the polymerase enzyme (e.g.,MgCl₂ and/or KCl).

The term “primer” as used herein is known to those skilled in the artand refers to oligomeric compounds, primarily to oligonucleotides butalso to modified oligonucleotides that are able to “prime” DNA synthesisby a template-dependent DNA polymerase, i.e., the 3′-end of the, e.g.,oligonucleotide provides a free 3′-OH group where further “nucleotides”may be attached by a template-dependent DNA polymerase establishing 3′to 5′ phosphodiester linkage whereby deoxynucleoside triphosphates areused and whereby pyrophosphate is released.

The term “hybridizing” refers to the annealing of one or more probes toan amplification product. “Hybridization conditions” typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

The term “5′ to 3′ nuclease activity” refers to an activity of a nucleicacid polymerase, typically associated with the nucleic acid strandsynthesis, whereby nucleotides are removed from the 5′ end of nucleicacid strand.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished, if necessary.

The term “complement thereof” refers to nucleic acid that is both thesame length as, and exactly complementary to, a given nucleic acid.

The term “extension” or “elongation” when used with respect to nucleicacids refers to when additional nucleotides (or other analogousmolecules) are incorporated into the nucleic acids. For example, anucleic acid is optionally extended by a nucleotide incorporatingbiocatalyst, such as a polymerase that typically adds nucleotides at the3′ terminal end of a nucleic acid.

The terms “identical” or percent “identity” in the context of two ormore nucleic acid sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same, when compared and aligned for maximumcorrespondence, e.g., as measured using one of the sequence comparisonalgorithms available to persons of skill or by visual inspection.Exemplary algorithms that are suitable for determining percent sequenceidentity and sequence similarity are the BLAST programs, which aredescribed in, e.g., Altschul et al. (1990) “Basic local alignment searchtool” J. Mol. Biol. 215:403-410, Gish et al. (1993) “Identification ofprotein coding regions by database similarity search” Nature Genet.3:266-272, Madden et al. (1996) “Applications of network BLAST server”Meth. Enzymol. 266:131-141, Altschul et al. (1997) “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs” NucleicAcids Res. 25:3389-3402, and Zhang et al. (1997) “PowerBLAST: A newnetwork BLAST application for interactive or automated sequence analysisand annotation” Genome Res. 7:649-656, which are each incorporatedherein by reference.

A “modified nucleotide” in the context of an oligonucleotide refers toan alteration in which at least one nucleotide of the oligonucleotidesequence is replaced by a different nucleotide that provides a desiredproperty to the oligonucleotide. Exemplary modified nucleotides that canbe substituted in the oligonucleotides described herein include, e.g., at-butyl benzyl, a C5-methyl-dC, a C5-ethyl-dC, a C5-methyl-dU, aC5-ethyl-dU, a 2,6-diaminopurine, a C5-propynyl-dC, a C5-propynyl-dU, aC7-propynyl-dA, a C7-propynyl-dG, a C5-propargylamino-dC, aC5-propargylamino-dU, a C7-propargylamino-dA, a C7-propargylamino-dG, a7-deaza-2-deoxyxanthosine, a pyrazolopyrimidine analog, a pseudo-dU, anitro pyrrole, a nitro indole, 2′-O-methyl ribo-U, 2′-O-methyl ribo-C,an N4-ethyl-dC, an N6-methyl-dA, a 5-propynyl dU, a 5-propynyl dC,7-deaza-deoxyguanosine (deaza G (u-deaza)) and the like. Many othermodified nucleotides that can be substituted in the oligonucleotides arereferred to herein or are otherwise known in the art. In certainembodiments, modified nucleotide substitutions modify meltingtemperatures (Tm) of the oligonucleotides relative to the meltingtemperatures of corresponding unmodified oligonucleotides. To furtherillustrate, certain modified nucleotide substitutions can reducenon-specific nucleic acid amplification (e.g., minimize primer dimerformation or the like), increase the yield of an intended targetamplicon, and/or the like in some embodiments. Examples of these typesof nucleic acid modifications are described in, e.g., U.S. Pat. No.6,001,611, which is incorporated herein by reference. Other modifiednucleotide substitutions may alter the stability of the oligonucleotide,or provide other desirable features.

Detection/Quantitation of BK Virus Target Nucleic Acid

The present disclosure provides methods to detect BK virus byamplifying, for example, a portion of the BK virus nucleic acidsequence. Specifically, primers and probes to amplify and detect and/orquantitate BK virus nucleic acid molecule targets are provided by theembodiments in the present disclosure.

For detection and/or quantitation of BK virus, primers and probes toamplify and detect/quantitate the BK virus are provided. BK virusnucleic acids other than those exemplified herein can also be used todetect BK virus in a sample. For example, functional variants can beevaluated for specificity and/or sensitivity by those of skill in theart using routine methods. Representative functional variants caninclude, e.g., one or more deletions, insertions, and/or substitutionsin the BK virus nucleic acids disclosed herein.

More specifically, embodiments of the oligonucleotides each include anucleic acid with a sequence selected from SEQ ID NOs:1-11, asubstantially identical variant thereof in which the variant has atleast, e.g., 80%, 90%, or 95% sequence identity to one of SEQ IDNOs:1-11, or a complement of SEQ ID NOs:1-11 and the variant.

TABLE 1  BK Virus Oligonucleotides SEQ Oligo ID Type NO: SequenceModifications Forward 1 CCTAACTCCTCAAACATATGC J: t-Butyl  Primer TGTJBenzyl-dA Reverse 2 ACAGTGGAAACTTTGTGATCC J: t-Butyl  Primer CJBenzyl-dA Probe 3 HATTGCQTGGTGCTCCTGZGG H: HEX-Thr CTATTGCTP Z: 7-deaza-deoxyguanosine Q: BHQ-2 P: Phosphate Forward 4 GGCTATAGCTGCTATAGGCCTJ: t-Butyl  Primer AJ Benzyl-dA Reverse 5 AGTAACAGTTTGAATTAAAGCK: t-Butyl  Primer AGCAAAK Benzyl-dC Forward 6 AGAGGAAAATCAGCACAAACCK: t-Butyl  Primer TK Benzyl-dC Reverse 7 CACCCTGACAAAGGGGGK K: t-Butyl Primer Benzyl-dC Probe 8 HTGAGCTAQCTCCAGGTTCCA H: HEX-ThrAAATCAGGCTGATGAP Q: BHQ-2 P: Phosphate Forward 9 CCTTTACATCCTGCTCCATTTJ: t-Butyl  Primer TTTTATJ Benzyl-dA Reverse 10 AGTGTAAGGAATTTCACCCTGJ: t-Butyl  Primer ACJ Benzyl-dA Probe 11 HAGTATTCQATTCTCTTCATTH: HEX-Thr TTATCCTCGTCGCCCCCTTP Q: BHQ-2 P: Phosphate

In one embodiment, the above described sets of BK virus primers andprobes are used in order to provide for detection of BK virus in abiological sample suspected of containing BK virus (Table 1). The setsof primers and probes may comprise or consist of the primers and probesspecific for the BK virus nucleic acid sequences, comprising orconsisting of the nucleic acid sequences of SEQ ID NOs:1-11. In anotherembodiment, the primers and probes for the BK virus target comprise orconsist of a functionally active variant of any of the primers andprobes of SEQ ID NOs:1-11.

A functionally active variant of any of the primers and/or probes of SEQID NOs:1-11 may be identified by using the primers and/or probes in thedisclosed methods. A functionally active variant of a primer and/orprobe of any of the SEQ ID NOs:1-11 pertains to a primer and/or probewhich provide a similar or higher specificity and sensitivity in thedescribed method or kit as compared to the respective sequence of SEQ IDNOs:1-11.

The variant may, e.g., vary from the sequence of SEQ ID NOs:1-11 by oneor more nucleotide additions, deletions or substitutions such as one ormore nucleotide additions, deletions or substitutions at the 5′ endand/or the 3′ end of the respective sequence of SEQ ID NOs:1-11. Asdetailed above, a primer and/or probe may be chemically modified, i.e.,a primer and/or probe may comprise a modified nucleotide or anon-nucleotide compound. A probe (or a primer) is then a modifiedoligonucleotide. “Modified nucleotides” (or “nucleotide analogs”) differfrom a natural “nucleotide” by some modification but still consist of abase or base-like compound, a pentofuranosyl sugar or a pentofuranosylsugar-like compound, a phosphate portion or phosphate-like portion, orcombinations thereof. For example, a “label” may be attached to the baseportion of a “nucleotide” whereby a “modified nucleotide” is obtained. Anatural base in a “nucleotide” may also be replaced by, e.g., a7-desazapurine whereby a “modified nucleotide” is obtained as well. Theterms “modified nucleotide” or “nucleotide analog” are usedinterchangeably in the present application. A “modified nucleoside” (or“nucleoside analog”) differs from a natural nucleoside by somemodification in the manner as outlined above for a “modified nucleotide”(or a “nucleotide analog”).

Oligonucleotides including modified oligonucleotides and oligonucleotideanalogs that amplify a nucleic acid molecule encoding the BK virustarget, e.g., nucleic acids encoding alternative portions of BK viruscan be designed using, for example, a computer program such as OLIGO(Molecular Biology Insights Inc., Cascade, Colo.). Important featureswhen designing oligonucleotides to be used as amplification primersinclude, but are not limited to, an appropriate size amplificationproduct to facilitate detection (e.g., by electrophoresis), similarmelting temperatures for the members of a pair of primers, and thelength of each primer (i.e., the primers need to be long enough toanneal with sequence-specificity and to initiate synthesis but not solong that fidelity is reduced during oligonucleotide synthesis).Typically, oligonucleotide primers are 8 to 50 nucleotides in length(e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, or 50 nucleotides in length).

In addition to a set of primers, the methods may use one or more probesin order to detect the presence or absence of BK virus. The term “probe”refers to synthetically or biologically produced nucleic acids (DNA orRNA), which by design or selection, contain specific nucleotidesequences that allow them to hybridize under defined predeterminedstringencies specifically (i.e., preferentially) to “target nucleicacids”, in the present case to a BK virus (target) nucleic acid. A“probe” can be referred to as a “detection probe” meaning that itdetects the target nucleic acid.

In some embodiments, the described BK virus probes can be labeled withat least one fluorescent label. In one embodiment, the BK virus probescan be labeled with a donor fluorescent moiety, e.g., a fluorescent dye,and a corresponding acceptor moiety, e.g., a quencher. In oneembodiment, the probe comprises or consists of a fluorescent moiety andthe nucleic acid sequences comprise or consist of SEQ ID NO:3, 8 or 11.

Designing oligonucleotides to be used as probes can be performed in amanner similar to the design of primers. Embodiments may use a singleprobe or a pair of probes for detection of the amplification product.Depending on the embodiment, the probe(s) use may comprise at least onelabel and/or at least one quencher moiety. As with the primers, theprobes usually have similar melting temperatures, and the length of eachprobe must be sufficient for sequence-specific hybridization to occurbut not so long that fidelity is reduced during synthesis.Oligonucleotide probes are generally 15 to 40 (e.g., 16, 18, 20, 21, 22,23, 24, or 25) nucleotides in length.

Constructs can include vectors each containing one of BK virus primersand probes nucleic acid molecules (e.g., SEQ ID NOs:1, 2, 3, 4, 5, 6, 7,8, 9, 10, and 11). Constructs can be used, for example, as controltemplate nucleic acid molecules. Vectors suitable for use arecommercially available and/or produced by recombinant nucleic acidtechnology methods routine in the art. BK virus nucleic acid moleculescan be obtained, for example, by chemical synthesis, direct cloning fromBK virus, or by nucleic acid amplification.

Constructs suitable for use in the methods typically include, inaddition to the BK virus nucleic acid molecules (e.g., a nucleic acidmolecule that contains one or more sequences of SEQ ID NOs:1-11),sequences encoding a selectable marker (e.g., an antibiotic resistancegene) for selecting desired constructs and/or transformants, and anorigin of replication. The choice of vector systems usually depends uponseveral factors, including, but not limited to, the choice of hostcells, replication efficiency, selectability, inducibility, and the easeof recovery.

Constructs containing BK virus nucleic acid molecules can be propagatedin a host cell. As used herein, the term host cell is meant to includeprokaryotes and eukaryotes such as yeast, plant and animal cells.Prokaryotic hosts may include E. coli, Salmonella typhimurium, Serratiamarcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts suchas S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COScells or Chinese hamster ovary (CHO) cells, insect cells, and plantcells such as Arabidopsis thaliana and Nicotiana tabacum. A constructcan be introduced into a host cell using any of the techniques commonlyknown to those of ordinary skill in the art. For example, calciumphosphate precipitation, electroporation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer are commonmethods for introducing nucleic acids into host cells. In addition,naked DNA can be delivered directly to cells (see, e.g., U.S. Pat. Nos.5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in some embodiments include oligonucleotidescapable of acting as points of initiation of nucleic acid synthesiswithin the described BK virus nucleic acid sequences (e.g., SEQ ID NOs:1, 2, 7, 9, and 10). A primer can be purified from a restriction digestby conventional methods, or it can be produced synthetically. The primeris preferably single-stranded for maximum efficiency in amplification,but the primer can be double-stranded. Double-stranded primers are firstdenatured, i.e., treated to separate the strands. One method ofdenaturing double stranded nucleic acids is by heating.

If the template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min (e.g., 1 min to 2 min 30 sec, or 1.5min).

If the double-stranded template nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence. The temperature forannealing is usually from about 35° C. to about 65° C. (e.g., about 40°C. to about 60° C.; about 45° C. to about 50° C.). Annealing times canbe from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec;about 30 sec to about 40 sec). The reaction mixture is then adjusted toa temperature at which the activity of the polymerase is promoted oroptimized, i.e., a temperature sufficient for extension to occur fromthe annealed primer to generate products complementary to the templatenucleic acid. The temperature should be sufficient to synthesize anextension product from each primer that is annealed to a nucleic acidtemplate, but should not be so high as to denature an extension productfrom its complementary template (e.g., the temperature for extensiongenerally ranges from about 40° C. to about 80° C. (e.g., about 50° C.to about 70° C.; about 60° C.). Extension times can be from about 10 secto about 5 min (e.g., about 30 sec to about 4 min; about 1 min to about3 min; about 1 min 30 sec to about 2 min).

The genome of a retrovirus or RNA virus, is comprised of a ribonucleicacid, i.e., RNA. In such case, the template nucleic acid, RNA, mustfirst be transcribed into complementary DNA (cDNA) via the action of theenzyme reverse transcriptase. Reverse transcriptases use an RNA templateand a short primer complementary to the 3′ end of the RNA to directsynthesis of the first strand cDNA, which can then be used directly as atemplate for polymerase chain reaction.

PCR assays can employ BK virus nucleic acid such as RNA or DNA (cDNA).The template nucleic acid need not be purified; it may be a minorfraction of a complex mixture, such as BK virus nucleic acid containedin human cells. BK virus nucleic acid molecules may be extracted from abiological sample by routine techniques such as those described inDiagnostic Molecular Microbiology: Principles and Applications (Persinget al. (eds), 1993, American Society for Microbiology, Washington D.C.).Nucleic acids can be obtained from any number of sources, such asplasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

The oligonucleotide primers (e.g., SEQ ID NOs:1, 2, 4, 5, 6, 7, 9, and10) are combined with PCR reagents under reaction conditions that induceprimer extension. For example, chain extension reactions generallyinclude 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl₂, 0.001% (w/v)gelatin, 0.5-1.0 μg denatured template DNA, 50 pmoles of eacholigonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO). Thereactions usually contain 150 to 320 μM each of dATP, dCTP, dTTP, dGTP,or one or more analogs thereof.

The newly-synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target BK virus nucleic acid molecules. The limiting factors inthe reaction are the amounts of primers, thermostable enzyme, andnucleoside triphosphates present in the reaction. The cycling steps(i.e., denaturation, annealing, and extension) are preferably repeatedat least once. For use in detection, the number of cycling steps willdepend, e.g., on the nature of the sample. If the sample is a complexmixture of nucleic acids, more cycling steps will be required to amplifythe target sequence sufficient for detection. Generally, the cyclingsteps are repeated at least about 20 times, but may be repeated as manyas 40, 60, or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donorfluorescent moiety and a corresponding acceptor fluorescent moiety arepositioned within a certain distance of each other, energy transfertakes place between the two fluorescent moieties that can be visualizedor otherwise detected and/or quantitated. The donor typically transfersthe energy to the acceptor when the donor is excited by light radiationwith a suitable wavelength. The acceptor typically re-emits thetransferred energy in the form of light radiation with a differentwavelength. In certain systems, non-fluorescent energy can betransferred between donor and acceptor moieties, by way of biomoleculesthat include substantially non-fluorescent donor moieties (see, forexample, U.S. Pat. No. 7,741,467).

In one example, an oligonucleotide probe can contain a donor fluorescentmoiety (e.g., HEX) and a corresponding quencher (e.g., BlackHoleQuencher™ (BHQ) (such as BHQ-2)), which may or not be fluorescent, andwhich dissipates the transferred energy in a form other than light. Whenthe probe is intact, energy transfer typically occurs between the donorand acceptor moieties such that fluorescent emission from the donorfluorescent moiety is quenched the acceptor moiety. During an extensionstep of a polymerase chain reaction, a probe bound to an amplificationproduct is cleaved by the 5′ to 3′ nuclease activity of, e.g., a TaqPolymerase such that the fluorescent emission of the donor fluorescentmoiety is no longer quenched. Exemplary probes for this purpose aredescribed in, e.g., U.S. Pat. Nos. 5,210,015, 5,994,056, and 6,171,785.Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonlyused quenchers are DABCYL and TAMRA. Commonly used dark quenchersinclude BlackHole Quencher™ (BHQ) (such as BHQ2), (BiosearchTechnologies, Inc., Novato, Calif.), Iowa Black™, (Integrated DNA Tech.,Inc., Coralville, Iowa), BlackBerry™ Quencher 650 (BBQ-650), (Berry &Assoc., Dexter, Mich.).

In another example, two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the BK virus target nucleic acid sequence.Upon hybridization of the oligonucleotide probes to the amplificationproduct nucleic acid at the appropriate positions, a FRET signal isgenerated. Hybridization temperatures can range from about 35° C. toabout 65° C. for about 10 sec to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system, or afluorimeter. Excitation to initiate energy transfer, or to allow directdetection of a fluorophore, can be carried out with an argon ion laser,a high intensity mercury (Hg) arc lamp, a xenon lamp, a fiber opticlight source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptor moieties“corresponding” refers to an acceptor fluorescent moiety or a darkquencher having an absorbance spectrum that overlaps the emissionspectrum of the donor fluorescent moiety. The wavelength maximum of theemission spectrum of the acceptor fluorescent moiety should be at least100 nm greater than the wavelength maximum of the excitation spectrum ofthe donor fluorescent moiety. Accordingly, efficient non-radiativeenergy transfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Foerster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,helium-cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine×isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate, or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm can be the distance in Angstroms (Å) from the nucleotide baseto the fluorescent moiety. In general, a linker arm is from about 10 Åto about 25 Å. The linker arm may be of the kind described in WO84/03285. WO 84/03285 also discloses methods for attaching linker armsto a particular nucleotide base, and also for attaching fluorescentmoieties to a linker arm.

An acceptor fluorescent moiety, such as an LC Red 640, can be combinedwith an oligonucleotide that contains an amino linker (e.g., C6-aminophosphoramidites available from ABI (Foster City, Calif.) or GlenResearch (Sterling, Va.)) to produce, for example, LC Red 640-labeledoligonucleotide. Frequently used linkers to couple a donor fluorescentmoiety such as fluorescein to an oligonucleotide include thiourealinkers (FITC-derived, for example, fluorescein-CPG's from Glen Researchor ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPGs that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of BK Virus Amplified Product (Amplicon)

The present disclosure provides methods for detecting the presence orabsence of BK virus in a biological or non-biological sample. Methodsprovided avoid problems of sample contamination, false negatives, andfalse positives. The methods include performing at least one cyclingstep that includes amplifying a portion of BK virus target nucleic acidmolecules from a sample using one or more pairs of BK virus primers, anda FRET detecting step. Multiple cycling steps are performed, preferablyin a thermocycler. Methods can be performed using the BK virus primersand probes to detect the presence of BK virus, and the detection of BKvirus indicates the presence of BK virus in the sample.

As described herein, amplification products can be detected usinglabeled hybridization probes that take advantage of FRET technology. OneFRET format utilizes TaqMan® technology to detect the presence orabsence of an amplification product, and hence, the presence or absenceof BK virus. TaqMan® technology utilizes one single-strandedhybridization probe labeled with, e.g., one fluorescent dye (e.g., HEX)and one quencher (e.g., BHQ-2), which may or may not be fluorescent.When a first fluorescent moiety is excited with light of a suitablewavelength, the absorbed energy is transferred to a second fluorescentmoiety or a dark quencher according to the principles of FRET. Thesecond moiety is generally a quencher molecule. During the annealingstep of the PCR reaction, the labeled hybridization probe binds to thetarget DNA (i.e., the amplification product, or amplicon) and isdegraded by the 5′ to 3′ nuclease activity of, e.g., the Taq Polymeraseduring the subsequent elongation phase. As a result, the fluorescentmoiety and the quencher moiety become spatially separated from oneanother. As a consequence, upon excitation of the first fluorescentmoiety in the absence of the quencher, the fluorescence emission fromthe first fluorescent moiety can be detected. By way of example, an ABIPRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan®technology, and is suitable for performing the methods described hereinfor detecting the presence or absence of BK virus in the sample.

Molecular beacons in conjunction with FRET can also be used to detectthe presence of an amplification product using the real-time PCRmethods. Molecular beacon technology uses a hybridization probe labeledwith a first fluorescent moiety and a second fluorescent moiety. Thesecond fluorescent moiety is generally a quencher, and the fluorescentlabels are typically located at each end of the probe. Molecular beacontechnology uses a probe oligonucleotide having sequences that permitsecondary structure formation (e.g., a hairpin). As a result ofsecondary structure formation within the probe, both fluorescentmoieties are in spatial proximity when the probe is in solution. Afterhybridization to the target nucleic acids (i.e., amplificationproducts), the secondary structure of the probe is disrupted and thefluorescent moieties become separated from one another such that afterexcitation with light of a suitable wavelength, the emission of thefirst fluorescent moiety can be detected.

Another common format of FRET technology utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand are generally designed to hybridize in close proximity to each otherin a target DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler® Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler® instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of BK virus genomes). If amplification of BKvirus target nucleic acid occurs and an amplification product isproduced, the step of hybridizing results in a detectable signal basedupon FRET between the members of the pair of probes.

Generally, the presence of FRET indicates the presence of BK virus inthe sample, and the absence of FRET indicates the absence of BK virus inthe sample. Inadequate specimen collection, transportation delays,inappropriate transportation conditions, or use of certain collectionswabs (calcium alginate or aluminum shaft) are all conditions that canaffect the success and/or accuracy of a test result, however.

Representative biological samples that can be used in practicing themethods include, but are not limited to respiratory specimens, urine,fecal specimens, blood specimens, plasma, dermal swabs, nasal swabs,wound swabs, blood cultures, skin, and soft tissue infections.Collection and storage methods of biological samples are known to thoseof skill in the art. Biological samples can be processed (e.g., bynucleic acid extraction methods and/or kits known in the art) to releaseBK virus nucleic acid or in some cases, the biological sample can becontacted directly with the PCR reaction components and the appropriateoligonucleotides.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the BK virus probes from the BK virusamplification products can confirm the presence or absence of BK virusin the sample.

Within each thermocycler run, control samples can be cycled as well.Positive control samples can amplify target nucleic acid controltemplate (other than described amplification products of target genes)using, for example, control primers and control probes. Positive controlsamples can also amplify, for example, a plasmid construct containingthe target nucleic acid molecules. Such a plasmid control can beamplified internally (e.g., within the sample) or in a separate samplerun side-by-side with the patients' samples using the same primers andprobe as used for detection of the intended target. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Each thermocycler run can alsoinclude a negative control that, for example, lacks target template DNA.Negative control can measure contamination. This ensures that the systemand reagents would not give rise to a false positive signal. Therefore,control reactions can readily determine, for example, the ability ofprimers to anneal with sequence-specificity and to initiate elongation,as well as the ability of probes to hybridize with sequence-specificityand for FRET to occur.

In an embodiment, the methods include steps to avoid contamination. Forexample, an enzymatic method utilizing uracil-DNA glycosylase isdescribed in U.S. Pat. Nos. 5,035,996, 5,683,896 and 5,945,313 to reduceor eliminate contamination between one thermocycler run and the next.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods. In one embodiment, a LightCycler® instrument isused. The following patent applications describe real-time PCR as usedin the LightCycler® technology: WO 97/46707, WO 97/46714, and WO97/46712.

The LightCycler® can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

One of skill in the art would appreciate that other nucleic acid- orsignal-amplification methods may also be employed. Examples of suchmethods include, without limitation, branched DNA signal amplification,loop-mediated isothermal amplification (LAMP), nucleic acidsequence-based amplification (NASBA), self-sustained sequencereplication (3 SR), strand displacement amplification (SDA), or smartamplification process version 2 (SMAP 2).

It is understood that the embodiments of the present disclosure are notlimited by the configuration of one or more commercially availableinstruments.

Articles of Manufacture/Kits

Embodiments of the present disclosure further provide for articles ofmanufacture or kits to detect BK virus. An article of manufacture caninclude primers and probes used to detect the BK virus gene target,together with suitable packaging materials. Representative primers andprobes for detection of BK virus are capable of hybridizing to BK virustarget nucleic acid molecules. In addition, the kits may also includesuitably packaged reagents and materials needed for DNA immobilization,hybridization, and detection, such solid supports, buffers, enzymes, andDNA standards. Methods of designing primers and probes are disclosedherein, and representative examples of primers and probes that amplifyand hybridize to BK virus target nucleic acid molecules are provided.

Articles of manufacture can also include one or more fluorescentmoieties for labeling the probes or, alternatively, the probes suppliedwith the kit can be labeled. For example, an article of manufacture mayinclude a donor and/or an acceptor fluorescent moiety for labeling theBK virus probes. Examples of suitable FRET donor fluorescent moietiesand corresponding acceptor fluorescent moieties are provided above.

Articles of manufacture can also contain a package insert or packagelabel having instructions thereon for using the BK virus primers andprobes to detect BK virus in a sample. Articles of manufacture mayadditionally include reagents for carrying out the methods disclosedherein (e.g., buffers, polymerase enzymes, co-factors, or agents toprevent contamination). Such reagents may be specific for one of thecommercially available instruments described herein.

Embodiments of the present disclosure also provide for a set of primersand one or more detectable probes for the detection of BK virus in asample.

Embodiments of the present disclosure will be further described in thefollowing examples, which do not limit the scope of the inventiondescribed in the claims.

EXAMPLES

The following examples and figures are provided to aid the understandingof the subject matter, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

The targeted region of the BK virus genome was the VP2 region of the BKvirus genome. All nucleic acid sequences were aligned and all primersand probes were considered and scored for their predicted inclusivityfor all known BK virus isolates and other properties.

Example 1: Specificity of Real-Time BK Virus PCR (SEQ ID NOs:3-5)

BK virus genomic DNA samples were used for a singleplex real-time PCRassay at a concentration of 4.0×10⁵ BK virus genome/μl. Reagents usedinclude Cobas® 6800/8800 generic PCR Master Mix, with the profile andconditions for use with the Cobas® 6800/8800, and using TaqMan®amplification and detection technology. The final concentration ofoligonucleotides in the master mix ranged from 0.10-0.40 μM. The Cobas®6800/8800 PCR Profile employed is depicted in Table 2, below:

TABLE 2 cobas ® 6800/8800 PCR Profile Target Hold time Step Cycles (°C.) (hh:mm:ss) Ramp Pre-PCR 1 50 00:02:00 4.4 94 00:00:05 4.4 5500:02:00 2.2 60 00:06:00 4.4 65 00:04:00 4.4 1. Meas 5 95 00:00:05 4.455 00:00:30 2.2 2. Meas 45 91 00:00:05 4.4 58 00:00:25 2.2 Post 1 4000:02:00 2.2

The oligonucleotides specific for BK virus used for the real-time PCRassay were SEQ ID NO:4 for the forward primer, SEQ ID NO:5 for thereverse primer, and SEQ ID NO:3 for the probe. These oligonucleotidestarget the VP2 region of the BK virus genome. The probe used in theseexamples is a TaqMan® probe.

The results of the real-time BK virus assay are shown in FIG. 1 , whichshows real-time PCR growth curves. Additionally, real-time PCR studieswere conducted in the presence of genomic DNA from other viruses, toconfirm specificity of the BK virus and lack of cross reactivity. Theresults, shown in FIG. 2 , demonstrate that the BK virus primers (SEQ IDNOs:4 and 5) and probe (SEQ ID NO:3) are specific for BK virus and donot cross react with genomic DNA from other viral samples (tested at1×10⁸ copies/μl) from Epstein Barr Virus (EBV), Herpes Simplex Virus-1(HSV-1), HSV-2, or cytomegalovirus (CMV). No signal was observed for thegenomic DNA samples, other than BK virus.

Thus, these results demonstrate that the primers and probes (SEQ IDNOs:3-5) amplify and detect the presence of BK virus specifically in areal-time PCR assay.

Example 2: Sensitivity of Real-Time BK Virus PCR (SEQ ID NOs:1-3)

BK virus genomic DNA samples were used for a real-time PCR assay at avarying concentrations, with a dilution panel including 1×10¹genomes/μl, 1×10² genomes/μl, 1×10³ genomes/μl, 1×10⁴ genomes/μl, 1×10⁵genomes/μl, and 4×10⁵ genomes/μl. Reagents used include Cobas® 6800/8800generic PCR Master Mix, with the profile and conditions for use with theCobas® 6800/8800, and using TaqMan® amplification and detectiontechnology. The final concentration of oligonucleotides in the mastermix ranged from 0.10-0.40 μM. The Cobas® 6800/8800 PCR Profile employedis depicted in Table 2, above.

The primers specific for BK virus used for this real-time PCR assay weredifferent than the ones employed in Example 1. Here, theoligonucleotides were SEQ ID NO:1 for the forward primer, SEQ ID NO:2for the reverse primer, and SEQ ID NO:3 for the probe. Theseoligonucleotides target the VP2 region of the BK virus genome. The probeused in these examples is a TaqMan® probe.

The results of the real-time BK virus assay are shown in FIG. 3 , whichshows real-time PCR growth curves of the dilution panel. These resultsshow that the primers (SEQ ID NOs:1 and 2) and probe (SEQ ID NO:3)amplify and detect BK virus in a dose-dependent fashion.

Thus, these results demonstrate the sensitivity of the primers andprobes (SEQ ID NOs:1-3) in amplifying and detecting the presence of BKvirus in a real-time PCR assay. The results indicate that the primers(SEQ ID NOs:1 and 2) and probe (SEQ ID NO:3) can achieve a sensitivitydown to 1×10¹ genomes/μl.

Example 3: Sensitivity of Real-Time BK Virus PCR (SEQ ID NOs:6-8)

BK virus genomic DNA samples were used for a real-time PCR assay at avarying concentrations, with a dilution panel including 1×10¹genomes/μl, 1×10² genomes/μl, and 1×10³ genomes/μl. Reagents usedinclude Cobas® 6800/8800 generic PCR Master Mix, with the profile andconditions for use with the Cobas® 6800/8800, and using TaqMan®amplification and detection technology. The final concentration ofoligonucleotides in the master mix ranged from 0.10-0.40 μM. The Cobas®6800/8800 PCR Profile employed is depicted in Table 2, above.

The oligonucleotides specific for BK virus used for the real-time PCRassay were SEQ ID NO:6 for the forward primer, SEQ ID NO:7 for thereverse primer, and SEQ ID NO:8 for the probe. These oligonucleotidestarget the small t-antigen region of the BK virus genome. The probe usedin these examples is a TaqMan® probe.

The results of the real-time BK virus assay are shown in FIG. 4 , whichshows real-time PCR growth curves of the dilution panel. These resultsshow that the primers (SEQ ID NOs:6 and 7) and probe (SEQ ID NO:8)amplify and detect BK virus in a dose-dependent fashion.

Thus, these results demonstrate the sensitivity of the primers andprobes (SEQ ID NOs:6-8) in amplifying and detecting the presence of BKvirus in a real-time PCR assay. The results indicate that the primers(SEQ ID NOs:6 and 7) and probe (SEQ ID NO:8) can achieve a sensitivitydown to 1×10¹ genomes/μl.

Example 4: Sensitivity of Real-Time BK Virus PCR (SEQ ID NOs:9-11)

BK virus genomic DNA samples were used for a real-time PCR assay at avarying concentrations, with a dilution panel including 1×10¹genomes/μl, 1×10² genomes/μl, and 1×10³ genomes/μl. Reagents usedinclude Cobas® 6800/8800 generic PCR Master Mix, with the profile andconditions for use with the Cobas® 6800/8800, and using TaqMan®amplification and detection technology. The final concentration ofoligonucleotides in the master mix ranged from 0.10-0.40 μM. The Cobas®6800/8800 PCR Profile employed is depicted in Table 2, above.

The oligonucleotides specific for BK virus used for the real-time PCRassay were SEQ ID NO:9 for the forward primer, SEQ ID NO:10 for thereverse primer, and SEQ ID NO:11 for the probe. These oligonucleotidestarget the small t-antigen region of the BK virus genome. The probe usedin these examples is a TaqMan® probe.

The results of the real-time BK virus assay are shown in FIG. 5 , whichshows real-time PCR growth curves of the dilution panel. These resultsshow that the primers (SEQ ID NOs:9 and 10) and probe (SEQ ID NO:11)amplify and detect BK virus in a dose-dependent fashion.

Example 5: Dual Target Multiplex Real-Time BK Virus PCR (SEQ ID NOs:3-5and SEQ ID NOs:6-8)

The oligonucleotides were tested in a multiplex real-time PCR assay,such that two targets (i.e., “dual target”) were tested (i.e., VP2 andsmall t-antigen). In this test, the BK virus standards were obtainedfrom the Exact Diagnostics BKV Verification Panel (Exact Diagnostics(EDX), Catalog Number BKVP100). The EDX BKV Verification Panel is astandard useful in a number of molecular assays, calibrated against the1^(st) WHO International Standard for BK Virus. The EDX BKV VerificationPanel includes whole intact virus and is used to measure the presence(qualitative and quantitative) of polyomavirus BK virus DNA. Thestandards were formulated in negative human plasma and spiked intospecimen diluent and was extracted using Cobas® 6800/8800 samplepreparation workflows. The eluates were run on the LightCycler® 480Instrument II and tested with dual target mastermix. The BK virusstandards were at a concentration ranging from 0.2 IU/r×n to 2×10⁴IU/r×n

The oligonucleotides specific for BK virus used for this multiplexreal-time PCR assay were one oligonucleotide set targeting the VP2region (SEQ ID NOs:3-5) and one oligonucleotide set targeting the smallt-antigen (SEQ ID NOs:6-8). The probe used in these examples is aTaqMan® probe.

The results of the real-time BK virus assay are shown in FIG. 6 , whichshows real-time PCR growth curves of the dilution panel. These resultsshow that the two sets of oligonucleotides (SEQ ID NOs:3-5 and SEQ IDNOs:6-8) are able to amplify and detect BK virus in a multiplex settingand in a dose-dependent fashion.

Example 6: Dual-Target Multiplex Real-Time BK Virus PCR (SEQ ID NOs:3-5and SEQ ID NOs:9-11)

The oligonucleotides were tested in a multiplex real-time PCR assay,such that two targets (i.e., “dual target”) were tested (i.e., VP2 andsmall t-antigen). In this test, the BK virus standards were obtainedfrom the Exact Diagnostics BKV Verification Panel (Exact Diagnostics(EDX), Catalog Number BKVP100). The EDX BKV Verification Panel is astandard useful in a number of molecular assays, calibrated against the1^(st) WHO International Standard for BK Virus. The EDX BKV VerificationPanel includes whole intact virus and is used to measure the presence(qualitative and quantitative) of polyomavirus BK virus DNA. Thestandards were formulated in negative human plasma and spiked intospecimen diluent and was extracted using Cobas® 6800/8800 samplepreparation workflows. The eluates were run on the LightCycler® 480Instrument II and tested with dual target mastermix. The BK virusstandards were at a concentration ranging from 2 IU/r×n to 2×10³ IU/r×n

The oligonucleotides specific for BK virus used for this multiplexreal-time PCR assay were one oligonucleotide set targeting the VP2region (SEQ ID NOs:3-5) and one oligonucleotide set targeting the smallt-antigen (SEQ ID NOs:9-11). The probe used in these examples is aTaqMan® probe.

The results of the real-time BK virus assay are shown in FIG. 7 , whichshows real-time PCR growth curves of the dilution panel. These resultsshow that the two sets of oligonucleotides (SEQ ID NOs:3-5 and SEQ IDNOs:9-11) are able to amplify and detect BK virus in a multiplex settingand in a dose-dependent fashion.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

What is claimed:
 1. A method for simultaneously detecting two targetnucleic acids of BK virus in a biological sample, wherein the two targetnucleic acids of BK virus comprise a first target nucleic acid of BKvirus and a second target nucleic acid of BK virus, the methodcomprising: (a) performing an amplification step comprising contactingthe biological sample with two sets of primers to produce one or moreamplification products, wherein the two sets of primers comprise a firstset of primers for detecting the first target nucleic acid of BK virusand a second set of primers for detecting the second target nucleic acidof BK virus, wherein if the first target nucleic acid of BK virus ispresent in the biological sample, then a first amplification product isproduced, and wherein if the second target nucleic acid of BK virus ispresent in the biological sample, then a second amplification product isproduced; (b) performing a hybridization step, comprising contacting thefirst amplification product with a first probe and contacting the secondamplification product with a second probe; and (c) performing adetection step, comprising detecting the presence or absence of the oneor more amplification products, wherein the presence of the firstamplification product is indicative of the presence of the first targetnucleic acid of BK virus in the biological sample and the absence of thefirst amplification product is indicative of the absence of the firsttarget nucleic acid of BK virus in the biological sample, and whereinthe presence of the second amplification product is indicative of thepresence of the second target nucleic acid of BK virus in the biologicalsample, and the absence of the second amplification product isindicative of the absence of the second target nucleic acid of BK virusin the biological sample; and wherein: (i) the first set of primerscomprises a first primer consisting of a nucleic acid sequence of SEQ IDNO:4, or a complement thereof, and a second primer consisting of anucleic acid sequence of SEQ ID NO:5, or a complement thereof; and thefirst probe consists of a nucleic acid sequence of SEQ ID NO:3, or acomplement thereof; and (ii) the second set of primers comprises a firstprimer consisting of a nucleic acid sequence of SEQ ID NO:6, orcomplements thereof, and a second primer consisting of a nucleic acidsequence of SEQ ID NO:7, or a complement thereof; and the second probeconsisting of a nucleic acid sequence of SEQ ID NO:8, or a complementthereof.
 2. The method of claim 1, wherein the biological sample isplasma.
 3. A method for simultaneously detecting two different targetnucleic acids of BK virus in a biological sample, wherein the twodifferent target nucleic acids of BK virus comprise: (1) a first targetnucleic acid of BK virus comprising a target nucleic acid from the viralcapsid protein 2 (VP2) region of the BK virus genome, and (2) a secondtarget nucleic acid of BK virus comprising a target nucleic acid fromthe small t antigen region of the BK virus genome, the methodcomprising: (a) performing an amplification step comprising contactingthe biological sample with two sets of primers, wherein the two sets ofprimers comprise a first set of primers and a second set of primers,wherein the first set of primers produces an amplification product ofthe target nucleic acid from the VP2 region of the BK virus genome, ifthe target nucleic acid from the VP2 region of the BK virus genome ispresent in the biological sample, and wherein the second set of primersproduces an amplification product of the target nucleic acid from thesmall t antigen region of the BK virus genome, if the target nucleicacid from the small t antigen region of the BK virus genome is presentin the biological sample; (b) performing a hybridization step,comprising contacting the amplification product of the target nucleicacid from the VP2 region of the BK virus genome with a first probe, andcontacting the amplification product of the target nucleic acid from thesmall t antigen region of the BK virus genome with a second probe; and(c) performing a detection step, comprising detecting the presence orabsence of the amplification products, wherein the presence of theamplification product of the target nucleic acid from the VP2 region ofthe BK virus genome is indicative of the presence of the target nucleicacid from the VP2 region of the BK virus genome in the biologicalsample, and absence of the amplification product of the target nucleicacid from the VP2 region of the BK virus genome is indicative of theabsence of the target nucleic acid from the VP2 region of the BK virusgenome in the biological sample; and wherein the presence of theamplification product of the target region from the small t antigenregion of the BK virus genome is indicative of the presence of thetarget region from the small t antigen region of the BK virus genome inthe biological sample, and absence of the amplification product of thetarget region from the small t antigen region of the BK virus genome isindicative of the absence of the target region from the small t antigenregion of the BK virus genome in the biological sample; and wherein: (i)the first set of primers comprises a first primer consisting of anucleic acid sequence of SEQ ID NO:4, or a complement thereof, and asecond primer consisting of a nucleic acid sequence of SEQ ID NO:5, or acomplement thereof; and the first probe consists of a nucleic acidsequence of SEQ ID NO:3, or a complement thereof; and (ii) the secondset of primers comprises a first primer consisting of a nucleic acidsequence of SEQ ID NO:6, or complements thereof, and a second primerconsisting of a nucleic acid sequence of SEQ ID NO:7, or a complementthereof; and the second probe consists of a nucleic acid sequence of SEQID NO:8, or a complement thereof.
 4. The method of claim 3, wherein thebiological sample is plasma.